The increased availability and identification of genes from human and other genomes has led to an increased need for efficient expression of recombinant proteins. The expression of proteins in bacteria is by far the most widely used approach for the production of cloned genes. For many reasons, expression in bacteria is preferred to expression in eukazyotic cells. For example, bacteria are much easier to grow than eukaryozic cells. More specifically, the availability of a wealth of sophisticated molecular genetic tools and thousands of mutants make E. coil, as an expression host, extremely useful for protein production. However, the high-level production of functional proteins in E. coli., especially those from eukaryotic sources has often been difficult. Inefficient translation initiation is the most common reason for poor expression. See e.g. Schoner et. al., Methods Enzymol. 185:94–103 (1990).
Initiation sites where the enzyme binds to specific nucleouides sequences are known as promoters. By determining the nucleotide sequences of protected regions from numerous E. coli genes, a consensus sequence for the E. coli promoter has been identified. The most conserved sequence is a hexamer (−10 element) centered at about the −10 position from the transcription initiation site. The consensus sequence is TATAAT. Upstream sequences around −35 also have a region of sequence similarity, TTGACA (−35 element), which is most evident in efficient promoters. The distance separating the −35 and −10 elements is between 16 and 18 base pairs in 90% of promoters. The distance is critical for maintaining necessary structural conformation of the two sites for RNA polymerase binding.
Regulation of promoter regions in prokaryotic genes contain multiple binding sites for transcription factors. Interactions between and among regulatory proteins bound to promoter regions produce both positive and negative synergistic effects on gene expression. Regulation of lac operon of E. coli has been studied extensively and has become a model for negative control of gene expression at the level of transcription. (See, e.g., Muller-Hill B., Prog Biophys Mol. Biol., 30(2–3): 227–52, 1975; and Sauer R T, Structure 4(3): 219–22, 1996.) Transcription of the lac operon in Escherichia coli is repressed by the binding of Lac repressor (LacR) to lac operator O1, a pseudo-palindromic sequence centered 11 base pairs downstream of the transcription start. Repressors appear to inhibit transcription by either steric hindrance preventing the binding of DNA polymerase to the promoter, or by preventing the transition of the closed state of the promoter to the open state. The lac repressor exists as a tetramer, and full repression of the wild-type lac promoter by wild-type LacR requires the presence of at least two other operator sequences that must not only be in close proximity to the lac operator O1, i.e. located at base pair 401 and 92 for the auxiliary operators O2 and O3, respectively, but the operator sequences must also be present on the same side of the DNA helix. LacR mutants lacking the C-terminal heptad repeat are only capable of dimer formation, yet still repress, albeit at a much reduced level. Repression of the lac promoter by the mutants is comparable to repression by tetrameric LacR when both auxiliary operators are not present. For a general review, see, Muller-Hill B. Curr Opin Microbiol. 1(2): 145–51, 1998.
Ideal promoter systems for overproducing recombinant proteins in E. coli should: (1) provide strong expression of cloned genes; (2) function only under inducing conditions; and (3) have a simple method of induction so that large-scale operation is possible. However, while the available repertoire of E. coli expression systems usually produce high levels of the corresponding cloned gene products, in many cases synthesize substantial levels of cloned gene products in uninduced or repressed conditions. Generally, these systems include controllable expression vectors based on the strong inducible promoter, tac and T7. Both promoter systems have only one lac operon site to regulate the target gene expression. Full repression of these two strong promoters has been difficult to achieve. Furthermore, the leaky basal expression of some toxic proteins can have a detrimental impact on cell growth and viability.
Despite advances in the expression of recombinant proteins in bacterial hosts, there exists a need for improved methods for higher yields for protein production.